Exploring cell-types in stimulated data

The stimulated cells part of the Cano-Gamez et al. data contains T cells that were activated by anti-Cd3/CD28 beads. Cytokines were added to force the development of subtypes.

Annotation ‘cytokine.condition’ TCR activation / anti-CD3/anti-CD28 beads Added Cytokines
UNS No None
Th0 Yes None
Th2 Yes IL4, anti-IFN-\gamma
Th17 Yes IL-6, IL-23, IL-1\beta, TGF-\beta, anti-IL-4, anti-IFN- \gamma
iTreg Yes TGF-\beta, IL-2

Loading the necessary libraries

Load preprocessed scRNA-seq data
See notebook "Data preprocessing" for this analysis part"

Separate the data in stimulated and UNS cells

Dimension Reduction

PCA

No clear ellbow. Only small proportion of variance explained.

UMAP

Louvain Clustering

The Louvain algorthim was chosen for clustering as it was used by Cano-Gamez et al.

! Caution: Rerunning your code will change the cluster composition due to randomness of the algorthim !

Identified 17 louvain clusters, same number as in the paper by Cano-Gamez et al.

Explore composition of the clusters

All clusters are a mixture of cytokine cell types. Most of them with high proportion of Th17 and iTreg.

Rank genes

Use identified genes to annotate IFN high, HSP high and Mitotic clusters:

Cluster Annotation

Used marker genes

The marker genes from literature are based on the following website: https://www.biocompare.com/Editorial-Articles/569888-A-Guide-to-T-Cell-Markers/

T cell type / differentiation state Marker genes mentioned in Cano-Gamez et al.
Tn/Th2 GATA3, MAOA, LIMA1, MRPS26
Tn/Th17 TNFRSF8, PALLD, RORA
Tn/iTreg FOXP3, LMCD1, LGALS3, CCL5
Tn/Th17/iTreg IL2, DUSP2, TNF
Tm/Th17/iTreg CCL5, LGALS3, TNFRSF8, BACH2, BATF3, AHR, IL17F, CTLA4
central memory T cells (Tcm) PASK
effector memory T cells (Tem) IL7R, KLRB1, TNFSF13B
terminally differentiated effector cells (TEMRA) PRF1, CCL4, GZMA, GZMH
natural T regulatory cells (nTreg) FOXP3, CTLA4
T cell type / differentiation state Marker genes literature
T naive (Tn) CCR7
central memory T cells (Tcm) FAS, IL2RB, PRDM1
effector memory T cells (Tem) CXCR3, ITGAL, CCR5, TBX21

Map marker genes on clusters

Tn/Th2

-> cluster 4

Tn/Th17

-> cluster 15, cluster 1

Tn/iTreg

-> cluster 6

Tn/Th17/iTreg

-> cluster 15, cluster 1

Tm/Th17/iTreg

Tcm

Tem

TEMRA

nTreg

Add annotation to AnnData

Create annotation

The annotation is based on marker genes and composition of the clusters. Since an attempt was made to identify the same clusters as by Cano-Gamez et al., some annotations may have little correspondence with the observations made above.

Add annotation to data

Plotting

Annotation does not match the one in the paper (see: https://cytokines.cellgeni.sanger.ac.uk/simulated). Take the output from different factor analyis algorithms (Factor_Analysis_Methods.ipynb) for alternative clustering.

Save data